The germinal center (GC) response is central in generating long-lived humoral immunity and the basis for effective vaccination. GC B cells (GCBC) extensively remodel their transcription factor (TF) networks and gene expression. GCBC undergo V region somatic hypermutation that generates the substrate for a cyclic process of positive and negative selection in which surviving GCBC divide 3-4 times per day. Among the most pivotal questions in GC biology is how signals lead to positive selection of higher affinity B cells, either by entering a new cycle of cell division and/or rescue from cell death. How is selection in GC balanced against death and differentiation into one of two possible long-lived fates: memory B cell (MBC) and long-lived plasma cell (LLPC). Recent studies have identified certain putative surface markers as well as opposing and interacting transcription factor networks that may control these events. One important clue comes from our recent studies showing that the early GC reaction tends to spawn MBC while the late phase largely generates LLPC. In this proposal we will use novel approaches to ask how different signals and downstream pathways interact to determine GCBC fate (selection and MBC/PC differentiation and to elucidate precursor-product relationships of GCBC and progeny. In work supported by our first cycle of funding, we have been focusing on how Ag-dependent signals?either directly, via BCR, or indirectly, via recruitment of T cell help through antigen presentation?control the fate of GCBC during positive selection in the GC. We found that BCR and CD40 signaling are dramatically reprogrammed in GCBC compared to nave B cells (NBC), with signaling from both receptors being substantially yet selectively attenuated. In contrast to NBC, GCBC require both BCR and CD40 to ignite a positive selection signal, as read out by expression of c-MYC and generation of p-S6. These observations raise a number of outstanding questions. First, are IL-21 signals also interpreted differently by GCBC, as we found that IL21 and CD40 stimulation only synergistically induce c-MYC in GCBC? Second, are cytokine signals contributing to positive selection and determining how GCBC differentiate and if so, how? Mechanistically, since we know that signals act in concert and synergistically, we would like to understand how BCR, CD40 and cytokine signals interact, or ?crosstalk?. Elucidating mechanisms of signal reprogramming and crosstalk will reveal how signals to GCBC are integrated at the molecular level to determine appropriate responses of those cells. We will test a hypothetical model that posits that early in the reaction, Ag is abundant and BCR signals predominate, which favors MBC generation and GC maintenance, while at later stages IL-21 signals prevail, which favors plasma cell generation. To test this model we will investigate, in Aim 1, how IL-21R signals are rewired in GCBC; in Aim 2, how, mechanistically, BCR/CD40 and IL-21/CD40 signals differ in terms of gene expression, epigenetics and TF network remodeling; and finally in Aim 3, how selective BCR and T cell signals determine fate outcome in vitro and in vivo. These experiments are at the conceptual and technical forefront of B cell biology.